Compounds useful in pain management

ABSTRACT

Compounds of general formula I                    
     are disclosed and claimed in the present application, as well as their pharmaceutically acceptable salts, pharmaceutical compositions comprising the novel compounds and their use in therapy, in particular in the management of pain. Intermediates to the compounds of the formula I are also claimed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application represents U.S. national stage of internationalapplication PCT/SE99/01076 which has an international filing date ofJun. 16, 1999 and which was published in English under PCT Article 21(2)on Dec. 29, 1999. The international application claims priority toSwedish application 9802207-2, filed oil Jun. 22, 1998.

FIELD OF THE INVENTION

The present invention is related to novel compounds, to a process fortheir preparation, their use and pharmaceutical compositions comprisingthe novel compounds. Also intermediates to the compounds of the presentapplication are claimed. The novel compounds are useful in therapy, andin particular for the treatment of pain.

BACKGROUND AND PRIOR ART

The δ receptor has been identified as having a role in many bodilyfunctions such as circulatory and pain systems. Ligands for the δreceptor may therefore find potential use as analgesics, and/or asantihypertensive agents. Ligands for the δ receptor have also been shownto possess immunomodulatory activities.

The identification of at least three different populations of opioidreceptors (μ, δ and κ) is now well established and all three areapparent in both central and peripheral nervous systems of many speciesincluding man. Analgesia has been observed in various animal models whenone or more of these receptors has been activated.

With few exceptions, currently available selective opioid δ ligands arepeptidic in nature and are unsuitable for administration by systemicroutes. Some non-peptidic δ antagonists have been available for sometime (see Takemori and Portoghese, 1992, Ann. Rev. Pharmacol. Tox., 32:239-269. for review). These compounds, e.g. naltrindole, suffer fromrather poor (i.e., <10-fold) selectivity for the δ receptor vs. μreceptor binding and exhibit no analgesic activity, a fact whichunderscores the need for the development of highly selectivenon-peptidic δ ligands.

Thus, the problem underlying the present invention was to find newanalgesics having improved analgesic effects, but also with an improvedside-effect profile over current μ agonists and potential oral efficacy.

Analgesics that have been identified and are existing in the prior arthave many disadvantages in that they suffer from poor pharmacokineticsand are not analgesic when administered by systemic routes. Also, it hasbeen documented that preferred compounds, described within the priorart, show significant convulsive effects when administered systemically.

The problem mentioned above has now been solved by developing novelsubstituted phenyl compounds, as will be described below.

OUTLINE OF THE INVENTION

The novel compounds according to the present invention are defined bythe general formula I

wherein

R¹ is selected from anyone of

(i) a straight or branched C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl,where each alkyl, alkenyl or alkynyl may optionally be substituted byone or more aromatic or heteroaromatic substituents;

(ii) C₃-C₇ cycloalkyl optionally substituted by anyone of C₁-C₆ alkyl,C₁-C₆ alkoxy, or hydroxy;

(iii) hydrogen, halogen or C₁-C₆ alkoxy;

(iv) C₆-C₁₀ aryl;

(v) heteroaryl having from 5 to 10 atoms and the heteroatom beingselected from any of S, N and O; wherein the aryl and heteroaryl mayoptionally and independently be substituted by 1 or 2 substituents Ywherein each Y is as defined below;

Q is selected from any of CH₃;

 wherein

R², R³ and R⁴ is each and independently selected from any of

(i) C₆-C₁₀ aryl; or

(ii) heteroaryl having from 5 to 10 atoms and the heteroatom beingselected from any of S, N and O; and

 wherein the aryl and heteroaryl may optionally and independently besubstituted by 1 or 2 substituents Y where each Y is as defined below;

(iii) hydrogen;

(iv) a straight or branched C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl;

(v) saturated or unsaturated C₃-C₁₀ cycloalkyl, optionally andindependently substituted by one or more heteroaryl(s) having from 5 to10 atoms and the heteroatom(s) being selected from any of S, N and O andwherein the heteroaryl may optionally and independently be substitutedby 1 or 2 substituents Y wherein each Y is as defined below;

Y is each and independently selected from any of hydrogen, CH₃;—(CH₂)_(p1)CF₃; halogen; C₁-C₃ alkoxy; hydroxy; —NO₂; —OCF₃;—CONR^(a)R^(b); COOR^(a); —COR^(a); —(CH₂)_(p2)NR^(a)R^(b);—(CH₂)_(p3)CH₃, (CH₂)_(p4)SOR^(a)R^(b); —(CH₂)_(p5)SO₂R^(a);—(CH₂)_(p6)SO₂NR^(a); C₄-C₈(alkyl-cycloalkyl) wherein alkyl is C₁-C₂alkyl and cycloalkyl is C₃-C₆ cycloalkyl; 1 or 2 heteroaryl(s) havingfrom 5 to 10 atoms and the heteroatom(s) being selected from any of S, Nand O; and oxides such as N-oxides or sulfoxides; and wherein

 R^(a) and R^(b) is each and independently selected from hydrogen, abranched or straight C₁-C₆ alkyl, C₁-C₆ alkenyl, C₃-C₈ cycloalkyl; andwherein

 p1, p2, p3, p4, p5 and p6 is each and independently 0, 1 or 2.

Within the scope of the invention are also pharmaceutically acceptablesalts of the compounds of the formula I, as well as isomers, hydrates,isoforms and prodrugs thereof.

Preferred compounds according to the invention are compounds of theformula I wherein

Q is

 wherein

R² and R⁴ is each and independently selected from any of

(i) C₆-C₁₀ aryl; or

(ii) heteroaryl having from 5 to 10 atoms and the heteroatom beingselected from any of S, N and O; and

 wherein the aryl and heteroaryl may optionally and independently besubstituted by 1 or 2 substituents Y where each Y is as defined above;

(iii) a straight or branched C₁-C₆ alkyl or C₂-C₆ alkynyl;

(iv) saturated or unsaturated C₃-C₆ cycloalkyl, optionally andindependently substituted by one or more heteroaryl(s) having from 5 to10 atoms and the heteroatom(s) being selected from any of S, N and O andwherein the heteroaryl may optionally and independently be substitutedby 1 or 2 substituents Y wherein each Y is as defined above;

Particularly preferred compounds according to the invention arecompounds of the formula I wherein

R¹ is

(i) phenyl optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined above;

(ii) naphthyl optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined above;

(iii) heteroaryl having from 5 to 10 atoms and the heteroatom beingselected from any of S, N and O; and wherein the aryl and heteroaryl mayoptionally and independently be substituted by 1 or 2 substituents Ywhere each Y is as defined above;

Q is

wherein

R² is

(i) phenyl optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined above; or

(ii) naphthyl optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined above.

By “halogen” we mean chloro, fluoro, bromo and iodo.

By “aryl” we mean an aromatic ring having 6 or 10 carbon atoms, such asphenyl and naphthyl.

By “heteroaryl” we mean an aromatic ring in which one or more of thefrom 5-10 atoms in the ring are elements other than carbon, such as N, Sand O.

By “isomers” we mean compounds of the formula I, which differ by theposition of their functional group and/or orientation. By “orientation”we mean stereoisomers, diastereoisomers, regioisomers and enantiomers.

By “isoforms” we mean compounds of the formula I which differ in therelative physical arrangement of molecules by crystal lattice, such thatisoforms refer to various crystalline compounds and amorphous compounds.

By “prodrug” we mean pharmacologically acceptable derivatives, e.g.esters and amides, such that the resulting biotransformation product ofthe derivative is an active form of the drug. The reference by Goodmanand Gilmans, The Pharmacological basis of Therapeutics, 8th ed.,McGraw-Hill, Int. Ed. 1992, “Biotransformation of Drugs, p. 13-15,describing prodrugs generally, is hereby incorporated by reference.

The novel compounds of the present invention are useful in therapy,especially for the treatment of various pain conditions such as chronicpain, acute pain, cancer pain, pain caused by rheumatoid arthritis,migraine, visceral pain etc. This list should however not be interpretedas exhaustive.

Compounds of the invention are useful as immunomodulators, especiallyfor autoimmune diseases, such as arthritis, for skin grafts, organtransplants and similar surgical needs, for collagen diseases, variousallergies, for use as anti-tumour agents and anti viral agents.

Compounds of the invention are useful in disease states wheredegeneration-or dysfunction of opioid receptors is present or implicatedin that paradigm. This may involve the use of isotopically labelledversions of the compounds of the invention in diagnostic techniques andimaging applications such as positron emission tomography (PET).

Compounds of the invention are useful for the treatment of diarrhoea,depression, urinary incontinence, various mental illnesses, cough, lungoedema, various gastrointestinal disorders, spinal injury and drugaddiction, including the treatment of alcohol, nicotine, opioid andother drug abuse and for disorders of the sympathetic nervous system forexample hypertension.

Compounds of the invention are useful as an analgesic agent for useduring general anaesthesia and monitored anaesthesia care. Combinationsof agents with different properties are often used to achieve a balanceof effects needed to maintain the anaesthetic state (e.g. Amnesia,analgesia, muscle relaxation and sedation). Included in this combinationare inhaled anaesthetics, hypnotica, anxiolytics, neuromuscular blockersand opioids.

The compounds of the present invention in isotopically labelled form areuseful as a diagnostic agent.

Also within the scope of the invention is the use of any of thecompounds according to the formula I above, for the manufacture of amedicament for the treatment of any of the conditions discussed above.

A further aspect of the invention is a method for the treatment of asubject suffering from any of the conditions discussed above, whereby aneffective amount of a compound according to the formula I above, isadministered to a patient in need of such treatment.

The best mode of performing the invention known at present, is to usethe compounds according to Example 1 (compound 11), Example 8 (compound18), Example 9 (compound 19), Example 10 (compound 20) and Example 11(compound 21). The numbering of the compounds is in accordance with thenumbering in the Schemes presented in the following.

Methods of Preparation

The compounds of the present invention may be prepared by following thesynthetic routes described in Scheme 1 below.

General Procedure for the Preparation of 1,4 or 1,3-guanidinomethylAminomethyl Xylylene

As shown in Scheme 1 above, compounds of the formula I may be obtainedfrom commercially available bis-amino-xylylene (compound II). CompoundII is converted into mono-(diBoc)-guanidinomethyl derivative m using aprotected guanylating reagent such as1-H-pyrazole-1-(N,N-bis(tert-butoxycarbonylcarboxamidine) in an organicsolvent such as THF.

The secondary amine of the formula V may be generated using a reductiveamination step where the compound of the formula III is reacted with analdehyde (compound IV wherein R¹ is as defined in formula I above), inthe presence of an acid such as acetic acid or a Lewis acid such asZnCl₂ and in a protic solvent such as methanol or ethanol in thepresence of a reducing agent such as sodium cyanoborohydride.

Compounds of the formula VI may be obtained by performing an acylatingreaction where compound V is mixed with an acid chloride or otherappropriate acylating reagent such as an acid anhydride in a solventsuch as methylene chloride and in the presence of a tertiary amine asbase, such as triethylamine.

Finally, compounds of the formula I may be obtained by cleavage of theBoc protecting group with an acid such as aqueous hydrochloric acid orby using organic acid such as trifluoroacetic acid in a solvent such asmethylene chloride.

In the formulas of Scheme 1 above, R¹ and Q are as defined in formula Iabove.

EXAMPLES

The invention will now be described in more detail by way of thefollowing Examples, which are not to be construed as limiting theinvention in any way.

Step 1(a)

Preparation of 1-(diBoc)-guanidinomethyl-4-aminomethyl Benzene (compound2)

Part A

1-H-pyrazole-1-carboxamidine was prepared according to Bernatowiczet.al., J. Org. Chem. 1992, 57, pp.2497-2502, and protected withdi-tert-butyl dicarbonate to give1-H-pyrazole-1-N,N-bis(tert-butoxycarbonyl)carboxamidine according toDrake et.al, Synth. 1994. pp.579-582.

Part B

To a solution of p-xylylenediamine (30.8 g, 0.226 mol) in THF (300 mL)was added a solution of1-H-Pyrazole-1-(N,N-bis(tert-butoxycarbonyl)carboxamidine (35.0 g, 0.113mol) in THF (100 mL). The solution was stirred at room temperature for 3h. The solvent was removed under reduced pressure. Water was added tothe residue and the aqueous mixture was extracted with ethyl acetate.The organic layer was washed with brine, dried over MgSO₄ andconcentrated. The product (compound 2) was purified by columnchromatography on silica gel using a mixture of methylenechloride:methanol as the eluent to give 24.3 g (57% yield) of1-(diBoc)-guanidinomethyl-4-aminomethyl benzene.

¹H NMR (CDC13) δ 8.5 (broad s, 1H), 7.32 (s, 4H), 4.65 (d, 2H), 3.89 (s,2H), 1.5 (s, 9H), 1.48 (s, 9H).

Step 1(b)

Preparation of 1-(diBoc)-guanidinomethyl-3-aminomethyl Benzene (compound3)

1-(diBoc)-guanidinomethyl-3-aminomethyl benzene (compound 3) wasprepared in a similar fashion from m-xylylenediamine and of1-H-Pyrazole-1-(N,N-bis(tert-butoxycarbonyl)carboxamidine.

¹H NMR (CDC13) δ 8.52 (broad s, 1H), 7.28-7.08 (m, 4H), 4.56 (d, 2H),3.81 (s, 2H), 1.42 (s, 9H), 1.39 (s, 9H).

Step 2

Reductive Amination—preparation of1-(diBoc)-guanidinomethyl-4-[N-(benzyl)-aminomethyl]benzene (compound 3)

To a methanolic solution (15 ml) of compound 2 (322 mg, 0.85 mol) anddiphenylacetaldehyde (183.7 mg, 0.94 mol) was added zinc chloride (127.5mg, 0.94 mmol) and sodium cyanoborohydride (64.2 mg, 1.02 mol). Themixture was stirred over night under nitrogen, wherafter the mixture wasdiluted with saturated aqueous sodium bicarbonate and extracted withmethylene chloride. The organic phase was washed with brine, dried overMgSO₄ and concentrated. This crude product was further purified bysilica gel chromatography using CH₂Cl₂/MeOH (95:5) as the solvent, togive 145 mg of the pure desired product (compound 3).

Specific examples illustrating the preparation of secondary amines, i.e.intermediates of the general formula V, are provided in Table 1 below.

TABLE 1 Inter- mediate com- Intermediate compound pound of the generalfomula V of Scheme 1 no. and the chemical name thereforeCharacterization data 3

¹H NMR (CDCl₃) δ 8.5(broad s, 1H), 7.23-7.14(m, 14H), 4.58(d, 2H),4.18(t, 1H), 3.78(s, 2H), 3.22(d, 2H), 1.50(s, 9H), 1.47(s, 9H).MS(FAB+): 559(M + H), 359. 1-(diBoc)-guanidinomethyl-4-[N-(2,2-diphenylethyl)aminomethyl] benzene 4

¹H NMR (DMSO-d₆) δ 8.6(broad t, 1H), 7.42-7.1(m, 10H), 4.52(t, 2H),3.7(s, 2H), 3.2(s, 2H), 1.5(s, 9H), 1.45(s, 9H). MS(FAB+): 503(M + H),403, 303. 1-(diBoc)-guanidinomethyl-4-[N-(4- chlorobenzyl)aminomethyl]benzene 5

¹H NMR (CDCl₃) δ 7.2(m, 9H), 4.5(s, 2H), 4.15(t, 2H), 3.7(s, 2H), 1.5(s,18H). 1-(diBoc)-guanidinomethyl-4-[N- (benzyl)aminomethyl] benzene 6

¹H NMR (CDCl₃) δ 8.62(broad s, 1H), 7.48-7.1(m, 8H), 4.62(d, 2H),3.95(s, 2H), 3.85(s, 2H), 1.53(s, 9H), 1.5(s,9H).1-(diBoc)-guanidinomethyl-4-[N-(2- chlorobenzyl)aminomethyl] benzene 7

¹H NMR (CDCl₃) δ 8.56(broad t, 1H), 7.4-7.15(m, 8H), 4.6(d, 2H), 3.8(s,4H), 1.56(s, 9H), 1.52(s, 9H). MS(APCI): 503(M + H), 403, 303.1-(diBoc)-guanidinomethyl-4-[N-(3- chlorobenzyl)aminomethyl] benzene 8

MS(ES+): 559(M + H), 459, 359. 1-(diBoc)-guanidinomethyl-3-[N-(2,2-diphenylethyl)aminomethyl] benzene 9

¹H NMR (CDCl₃) δ 8.48(broad s, 1H), 7.3-6.96(m, 8H), 4.68-4.32(m, 4H),3.78-3.6(m, 2H). 1-(diBoc)-guanidinomethyl-3-[N-(4-chlorobenzyl)aminomethyl] benzene

Example 1

Preparation of 4-N-(benzoyl)-N-(2,2-diphenylethyl)aminomethyl-1-guanidinomethyl-p-xylylene (compound 11)

Compound 11 of this Example was prepared by following the reactionprocedure described in Scheme 2 below.

To a solution of compound 3 (145 mg, 0.26 mol) in methylene chloride (10ml) was added benzoyl chloride (2.96 mg, 0.52 mol) and triethylamine(52.52 mg, 0.52 mmol). The mixture was stirred at room temperature for 2h, washed with saturated NH₄Cl (aq) and brine, dried over MgSO₄ andconcentrated to give the crude product (compound 10). This crudecompound was used directly without purification for the preparation ofcompound 11. It was dissolved in dry methylene chloride (3 ml), 1.5 mlof TFA was added and the reaction mixture was stirred at roomtemperature for 1 hour. The excess of solvent and TFA was evaporated,the residue was purified by reverse phase preparative HPLC to give thepure desired product (100 mg, 84%) (compound 11).

¹H NMR (DMSO-d₆) δ 7.24-6.75 (m, 19H), 4.50-3.42 (m, 7H).

MS: 463.07 (M+H).

Examples 2-6

The following compounds were prepared by using the same procedure asdescribed in Example 1, but using the intermediate and the reagentindicated in Table 2 below.

TABLE 2 Physical Structure and Intermediate Charac- Ex chemical nameused Reagent terization 2

4 1-napthoyl chloride ¹H NMR (DMSO-d₆) δ 8.6(broad, 1H), 7.9(s, 1H),7.55-7.1(m, 14H), 4.6(broad, 4H), 4.3(d, 2H). (12)4-N-(1-Naphthoyl)-N-(4- chlorobenzyl)-aminomethyl-1-guanidinomethyl-p-xylene 3

3 2,6- dichloro- benzoyl- chloride ¹H NMR (DMSO-d₆) δ 7.5-7.1(m, 17H),4.55(t, 1H), 4.35(d, 2H), 4.1(d, 4H). (13) 4-N-(2,6-dichlorobenzoyl)-N-(2,2-diphenylethyl)- aminomethyl-1- guanidinomethyl-p-xylene 4

3 2,6- dimethoxy- benzoyl chloride ¹H NMR (DMSO-d₆) δ 7.5-7.2(m, 7H),7.05(d, 6H), 6.75(d, 2H), 6.55(d, 2H), 4.55(t, 1H), 4.40(m, 4H), 4.35(d,2H), 3.8(s, 6H). (14) 4-N-(2,6-dichlorobenzoyl)- N-(2,2-diphenylethyl)-aminomethyl-1-guanidino methyl-p-xylylene 5

8 2,6- dichloro- benzoyl- chloride ¹H NMR (DMSO-d₆) δ 8.0(broad, 1H),7.5-7.0(m, 17H), 4.7(t, 1H), 4.55(d, 1H), 4.45(d, 1H), 4.3-4.1(m, 4H).(15) 3-N-(2,6-dichlorobenzoyl)- N-(2,2-diphenylethyl)- aminomethyl-1-guanidinomethyl-m-xylylene 6

10 benzoyl chloride ¹H NMR (COCl₃) δ9.9(broad, 1H); 8.6(broad, 1H),.9-8.05(m, 4H); 7.55(m, 3H), 7.2-7.4(m, 10H), 4.6(broad, 4H), 4.4(d,2H). (16) 3-N-(2-naphtoyl)-N-(4- chlorobenzyl)-aminomethyl-1-guanidinomethyl-m-xylylene 7

5 isopropyl- phenyl sulphonyl- chloride ¹H NMR (DMSO-d₆) δ8.0(broad,1H), 7.9(d, 2H), 7.5(d, 2H), 7.3-7.0(m, 8H), 4.3(broad d, 6H), 3.05(m,1H), 1.25(d, 6H). MS(APCI): 485. (M + H). (17)4-N-(4-isopropylphenylsulfonyl)- N-(4-dichlorobenzyl)- aminomethyl-1-guanidinomethyl-p-xylylene

Examples 8-11

The following compounds were prepared by following the synthetic routedescribed in Scheme 3 below.

General Procedure

Compound 1 (commercially available) is converted intomono-(diBoc)-guanidinomethyl derivative2 using a protected guanylatingreagent such as1-H-pyrazole-1-(N,N-bis(tert-butoxycarbonyl)carboxamidine in an organicsolvent such as THF.

The secondary amine of the formula III may be generated using areductive amination step, where compound 2 is reacted with an aldehydeII in the presence of an acid such as acetic acid or a Lewis acid suchas ZnCl2, in a protic solvent such as methanol or ethanol in thepresence of a reducing agent such as sodium cyanobrohydride.

Compounds of the formula IV may be obtained by performing an acetylationusing compound III with the benzoylchloride in a solvent such asmethylene chloride and in the presence of a tertiary amine as base, suchas triethylamine.

Finally, a compound of formula V may be obtained by cleavage of the Bocprotecting group with an acide such as aqueous hydrochloric acid or byusing organic acid such as trifluoroacetic acid in a solvent such as amethylene chloride.

Example 8

Preparation ofN-[4-({[amino(imino)methyl]amino}methyl)benzyl-N-(1-naphtylmethyl)benzamide(compound 18)

To a methanolic solution(15 ml) of compound 2(100 mg, 0.264 mmol) and1-Naphathylaldehyde(41 mg, 0.264 mmol) was added zinc chloride(35.97 mg,0.264 mmol) and sodium cyanoborohydride(18.25 mg, 0.29 mmol). Themixture was stirred over night under nitrogen. Then the mixture wasdiluted with saturated aqueous sodium bicarbonate and extracted withmethylene chloride. The organic phase was washed with brine, dried overMgSO₄ and concentrated. This crude product was further purified bysilica gel chromatography using CH₂Cl₂/MeOH (95:5) as the solvent togive 50 mg of the pure desired product (compound 3 of formula III).

Preparation of Compound 4 & 5

To a solution of compound 3(50 mg, 0.096 mmol) in methylene chloride(10ml) was added benzoylchloride (27 mg, 0.192 mmol) and triethylamine(19mg, 0.192 mmol). The mixture was stirred at room temperature for 2 h.,washed with saturated NH₄Cl aqueous solution and brine, dried over MgSO₄and concentrated to give the crude product(compound 4 of formula IV).This crude compound was used directly without purification for thepreparation of compound 5 of formula V. It was dissolved in drymethylene chloride(3 ml), 1.5 ml of TFA was added and the reactionmixture was stirred at rt for 1 h. The excess of solvent and TFA wasevaporated, the residue was purified by reverse phase preparative HPLCto give the pure desired product (47 mg).

HPLC: >99% (215 nm), >99% (254 nm)

M.S.: Calc. 423.53 (MH+), Observed 423.50 (MH+)

Example 9

Preparation ofN-[4-({[amino(imino)methyl]amino}methyl)benzyl-N-(3-methyl-1-benzothiophen-2-yl)methyl)benzamide(compound 19)

Compound 19 was prepared by following the synthetic procedure asdescribed for Example 8 (compound 18). The reagent used was benzoylchloride, and the intermediate used was

prepared in a way analogous to intermediate no. 4 above, but using3-methylthiophene-2-carboxaldehyde instead of 4-chlorobenzaldehyde.

Physical Characterization for Compound 19:

HPLC: >99% (215 nm), >99% (254 nm)

M.S.: Calc. 443.59 (MH+), Observed 443.45 (MH+)

Example 10

Preparation ofN-[4-({[amino(imino)methyl]amino}methyl)benzyl-N-(3-fluorobenzyl)benzamide(compound 20)

Compound 20 was prepared by following the synthetic procedure asdescribed for Example 8 (compound 18). The reagent used was benzoylchloride, and the intermediate used was

prepared in a way analogous to intermediate no. 4 above, but using3-fluoro benzaldehyde instead of 4-chlorobenzaldehyde.

Physical Characterization for Compound 20:

HPLC: >99% (215 nm), >99% (254 nm)

M.S.: Calc. 391.46 (MH+), Observed 391.44 (MH+)

Example 11

Preparation ofN-[4-({[amino(imino)methyl]amino}methyl)benzyl-N-(2,6-dichlorobenzyl)benzamide(compound 21)

Compound 21 was prepared by following the synthetic procedure asdescribed for Example 8 (compound 18). The reagent used was benzoylchloride, and the intermediate used was

prepared in a way analogous to intermediate no. 4 above, but using2,6-dichloro benzaldehyde instead of 4-chiorobenzaldehyde.

Physical Characterization for Compound 21:

HPLC: >99% (215 nm), >99% (254 nm)

M.S.: Calc. 442.36 (MH+), Observed 441.37, 443.39 (M+2)

Pharmaceutical Compositions

The novel compounds according to the present invention may beadministered orally, intramuscularly, subcutaneously, topically,intranasally, intraperitoneally, intrathoracially, intravenously,epidurally, intrathecally, intracerebroventricularly and by injectioninto the joints.

A preferred route of administration is orally, intravenously orintramuscularly.

The dosage will depend on the route of administration, the severity ofthe disease, age and weight of the patient and other factors normallyconsidered by the attending physician, when determining the individualregimen and dosage level as the most appropriate for a particularpatient.

For preparing pharmaceutical compositions from the compounds of thisinvention, inert, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,dispersible granules, capsules, cachets, and suppositories.

A solid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be anencapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

For preparing suppository compositions, a low-melting wax such as amixture of fatty acid glycerides and cocoa butter is first melted andthe active ingredient is dispersed therein by, for example, stirring.The molten homogeneous mixture is then poured into convenient sizedmolds and allowed to cool and solidify.

Suitable carriers are magnesium carbonate, magnesium stearate, talc,lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose,sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and thelike.

Pharmaceutically acceptable salts are acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium acetate, camsylate,carbonate, chloride, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, glucaptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,lactobionate, malate, maleate, mandelate mesylate, methylbromide,methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate(embonate), pantothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate,teoclate, triethiodide, benzathine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglurnine, procaine, aluminium,calcium, lithium, magnesium, potassium, sodium, and zinc.

Preferred pharmaceutically acceptable salts are the hydrochlorides,trifluoroacetates and bitartrates.

The term composition is intended to include the formulation of theactive component with encapsulating material as a carrier providing acapsule in which the active component (with or without other carriers)is surrounded by a carrier which is thus in association with it.Similarly, cachets are included.

Tablets, powders, cachets, and capsules can be used as solid dosageforms suitable for oral administration.

Liquid from compositions include solutions, suspensions, and emulsions.Sterile water or water-propylene glycol solutions of the activecompounds may be mentioned as an example of liquid preparations suitablefor parenteral administration. Liquid compositions can also beformulated in solution in aqueous polyethylene glycol solution.

Aqueous solutions for oral administration can be prepared by dissolvingthe active component in water and adding suitable colorants, flavoringagents, stabilizers, and thickening agents as desired. Aqueoussuspensions for oral use can be made by dispersing the finely dividedactive component in water together with a viscous material such asnatural synthetic gums, resins, methyl cellulose, sodium carboxymethylcellulose, and other suspending agents known to the pharmaceuticalformulation art.

Preferably the pharmaceutical compositions is in unit dosage form. Insuch form, the composition is divided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofthe preparations, for example, packeted tablets, capsules, and powdersin vials or ampoules. The unit dosage form can also be a capsule,cachet, or tablet itself, or it can be the appropriate number of any ofthese packaged forms.

BIOLOGICAL EVALUATION

A) IN VITRO MODEL

Cell Culture

Human 293S cells expressing cloned human μ, δ, and κ receptors andneomycin resistance were grown in suspension at 37° C. and 5% CO₂ inshaker flasks containing calcium-free DMEM10% FBS, 5% BCS, 0.1% PluronicF-68, and 600 μg/ml geneticin.

Membrane Preparation

Cells were pelleted and resuspended in lysis buffer (50 mM Tris, pH 7.0,2.5 mM EDTA, with PMSF added just prior to use to 0.1 mM from a 0.1 Mstock in ethanol), incubated on ice for 15 min, then homogenized with apolytron for 30 sec. The suspension was spun at 1000 g (max) for 10 minat 4° C. The supernatant was saved on ice and the pellets resuspendedand spun as before. The supernatants from both spins were combined andspun at 46,000 g(max) for 30 min. The pellets were resuspended in coldTris buffer (50 mM Tris/Cl, pH 7.0) and spun again. The final pelletswere resuspended in membrane buffer (50 mM Tris, 0.32 M sucrose, pH7.0). Aliquots (1 ml) in polypropylene tubes were frozen in dryice/ethanol and stored at −70° C. until use. The protein concentrationswere determined by a modified Lowry assay with SDS.

Binding Assays

Membranes were thawed at 37° C., cooled on ice, passed 3 times through a25-gauge needle, and diluted into binding buffer (50 mM Tris, 3 mMMgCl₂, 1 mg/ml BSA (Sigma A-7888), pH 7.4, which was stored at 4° C.after filtration through a 0.22 m filter, and to which had been freshlyadded 5 μg/ml aprotinin, 10 μM bestatin, 10 μM diprotin A, no DTT).Aliquots of 100 μl (for pg protein, see Table 1) were added to iced12×75 mm polypropylene tubes containing 100 μl of the appropriateradioligand (see Table 1) and 100 μl of test peptides at variousconcentrations. Total (TB) and nonspecific (NS) binding were determinedin the absence and presence of 10 μM naloxone respectively. The tubeswere vortexed and incubated at 25° C. for 60-75 min, after which timethe contents are rapidly vacuum-filtered and washed with about 12ml/tube iced wash buffer (50 mM Tris, pH 7.0, 3 MM MgCl₂) through GF/Bfilters (Whatman) presoaked for at least 2 h in 0.1% polyethyleneimine.The radioactivity (dpm) retained on the filters was measured with a betacounter after soaking the filters for at least 12 h in minivialscontaining 6-7 ml scintillation fluid. If the assay is set up in96-place deep well plates, the filtration is over 96-place PEI-soakedunifilters, which were washed with 3×1 ml wash buffer, and dried in anoven at 55° C. for 2 h. The filter plates were counted in a TopCount(Packard) after adding 50 μl MS-20 scintillation fluid/well.

Data Analysis

The specific binding (SB) was calculated as TB-NS, and the SB in thepresence of various test peptides was expressed as percentage of controlSB. Values of IC₅₀ and Hill coefficient (n_(H)) for ligands indisplacing specifically bound radioligand were calculated from logitplots or curve fitting programs such as Ligand, GraphPad Prism,SigmaPlot, or ReceptorFit. Values of K_(i) were calculated from theCheng-Prussoff equation. Mean±S.E.M. values of IC₅₀, K_(i) and n_(H)were reported for ligands tested in at least three displacement curves.

Receptor Saturation Experiments

Radioligand K_(δ) values were determined by performing the bindingassays on cell membranes with the appropriate radioligands atconcentrations ranging from 0.2 to 5 times the estimated K_(δ) (up to 10times if amounts of radioligand required are feasable). The specificradioligand binding was expressed as pmole/mg membrane protein. Valuesof K_(δ) and B_(max) from individual experiments were obtained fromnonlinear fits of specifically bound (B) vs. nM free (F) radioligandfrom individual according to a one-site model.

B) BIOLOGICAL MODEL (IN VIVO MODEL)

FREUND'S COMPLETE ADJUVANT (FCA), AND SCIATIC NERVE CUFF INDUCEDMECHANO-ALLODYNIA IN RAT

Animals

Male Sprague-Dawley rats (Charles River, St-Constant, Canada) weighing175-200 g at the time of surgery were used. They were housed in groupsof three in rooms thermostatically maintained at 20° C. with a 12:12 hrlight/dark cycle, and with free access to food and water. After arrival,the animals were allowed to acclimatize for at least 2 days beforesurgery. The experiments were approved by the appropriate MedicalEthical Committee for animal studies.

EXPERIMENTAL PROCEDURE

FREUND'S COMPLETE ADJUVANT

The rats were first anesthetized in a Halothane chamber after which 10μl of FCA was injected s.c. into the dorsal region of the left foot,between the second and third external digits. The animals were thenallowed to recover from anesthesia under observation in their home cage.

SCIATIC NERVE CUFF

The animals were prepared according to the method described by Mosconiand Kruger (1996). Rats were anesthetized with a mixture ofKetamine/Xylazine i.p. (2 ml/kg) and placed on their right side and anincision made over, and along the axis of, the lateral aspect of theleft femur. The muscles of the upper quadriceps were teased apart toreveal the sciatic nerve on which a plastic cuff (PE-60 tubing, 2 mmlong) was placed around. The wound was then closed in two layers with3-0 vicryl and silk sutures.

DETERMINATION OF MECHANO-ALLODYNIA USING VON FREY TESTING

Testing was performed between 08:00 and 16:00 h using the methoddescribed by Chaplan et al. (1994). Rats were placed in Plexiglas cageson top of a wire mesh bottom which allowed access to the paw, and wereleft to habituate for 10-15 min. The area tested was the mid-plantarleft hind paw, avoiding the less sensitive foot pads. The paw wastouched with a series of 8 Von Frey hairs with logarithmicallyincremental stiffness (0.41, 0.69, 1.20, 2.04, 3.63, 5.50, 8.51, and15.14 grams; Stoelting, Ill., USA). The von Frey hair was applied fromunderneath the mesh floor perpendicular to the plantar surface withsufficient force to cause a slight buckling against the paw, and heldfor approximately 6-8 seconds. A positive response was noted if the pawwas sharply withdrawn. Flinching immediately upon removal of the hairwas also considered a positive response. Ambulation was considered anambiguous response, and in such cases the stimulus was repeated.

TESTING PROTOCOL

The animals were tested on postoperative day 1 for the FCA-treated groupand on post-operative day 7 for the Sciatic Nerve Cuff group. The 50%withdrawal threshold was determined using the up-down method of Dixon(1980). Testing was started with the 2.04 g hair, in the middle of theseries. Stimuli were always presented in a consecutive way, whetherascending or descending. In the absence of a paw withdrawal response tothe initially selected hair, a stronger stimulus was presented; in theevent of paw withdrawal, the next weaker stimulus was chosen. Optimalthreshold calculation by this method requires 6 responses in theimmediate vicinity of the 50% threshold, and counting of these 6responses began when the first change in response occurred, e.g. thethreshold was first crossed. In cases where thresholds fell outside therange of stimuli, values of 15.14 (normal sensitivity) or 0.41(maximally allodynic) were respectively assigned. The resulting patternof positive and negative responses was tabulated using the convention,X=no withdrawal; O=withdrawal, and the 50% withdrawal threshold wasinterpolated using the formula:

50% g threshold=10^((Xf+kδ))/10,000

where Xf=value of the last von Frey hair used (log units); k=tabularvalue (from Chaplan et al. (1994)) for the pattern of positive/negativeresponses; and δ=mean difference between stimuli (log units). Hereδ=0.224.

Von Frey thresholds were converted to percent of maximum possible effect(% MPE), according to Chaplan et al. 1994. The following equation wasused to compute % MPE:${\% \quad {MPE}} = {\frac{{{Drug}\quad {treated}\quad {threshold}\quad (g)} - {{allodynia}\quad {threshold}\quad (g)}}{{{Control}\quad {threshold}\quad (g)} - {{allodynia}\quad {threshold}\quad (g)}} \times 100}$

ADMINISTRATION OF TEST SUBSTANCE

Rats were injected (subcutaneously, intraperitoneally, or orally) with atest substance prior to von Frey testing, the time betweenadministration of test compound and the von Frey test varied dependingupon the nature of the test compound.

What is claimed is:
 1. A compound according to formula I

wherein R¹ is selected from any one of (i) a straight or branched C₁-C₆alky, C₂-C₆ alkenyl or C₂-C₆ alkynyl, where each alkyl, alkenyl oralkynyl may optionally be substituted by one or more aromatic orheteroaromatic substituents; (ii) C₃-C₇ cycloalkyl optionallysubstituted by any one of C₁-C₆ alkyl, C₁-C₆ alkoxy, or hydroxy; (iii)hydrogen, halogen or C₁-C₆ alkoxy; (iv) C₆-C₁₀ aryl; (v) heteroarylhaving from 5 to 10 atoms, each heteroatom being selected from any of S,N and O; wherein the aryl and heteroaryl may optionally andindependently be substituted by 1 or 2 substituents Y wherein each Y isas defined below; Q is selected from any of CH₃;

 wherein R², R³ and R⁴ are each and independently selected from any of(i) C₆-C₁₀ aryl; or (ii) heteroaryl having from 5 to 10 atoms, eachheteroatom being selected from any of S, N and O; and  wherein the aryland heteroaryl may optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined below; (iii) hydrogen; (iv) astraight or branched C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl; (v)saturated or unsaturated C₃-C₁₀ cycloalkyl, optionally and independentlysubstituted by one or more heteroaryls having from 5 to 10 atoms, eachheteroatom being selected from any of S, N and O and wherein theheteroaryl may optionally and independently be substituted by 1 or 2substituents Y wherein each Y is as defined below; Y is each andindependently selected from any of hydrogen, CH₃; —(CH₂)_(p1)CF₃;halogen; C₁-C₃ alkoxy; hydroxy; —NO₂; —OCF₃; —CONR^(a)R^(b); —COOR^(a);—COR^(a); —(CH₂)_(p2)NR^(a)R^(b); —(CH₂)_(p3)CH₃;(CH₂)_(p4)SOR^(a)R^(b); —(CH₂)_(p5)SO₂R^(a); —(CH₂)_(p6)SO₂NR^(a);C₄-C₈(alkyl-cycloalkyl) wherein alkyl is C₁-C₂ alkyl and cycloalkyl isC₃-C₆ cycloalkyl; 1 or 2 heteroaryls having from 5 to 10 atoms, eachheteroatom being selected from any of S, N and O; and oxides; andwherein R^(a) and R^(b) are each and independently selected fromhydrogen, a branched or straight C₁-C₆ alkyl, C₁-C₆ alkenyl, C₃-C₈cycloalkyl; and wherein p1, p2, p3, p4, p5 and p6 are each andindependently 0, 1 or 2; or a pharmaceutically acceptable salt thereof.2. A compound according to formula I of claim 1, wherein Q is

 wherein R² and R⁴ is each and independently selected from any of (i)C₆-C₁₀ aryl; or (ii) heteroaryl having from 5 to 10 atoms and theheteroatom being selected from any of S, N and O; and  wherein the aryland heteroaryl may optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined in claim 1; (iii) a straightor branched C₁-C₆ alkyl or C₂-C₆ alkynyl; (iv) saturated or unsaturatedC₃-C₆ cycloalkyl, optionally and independently substituted by one ormore heteroaryl(s) having from 5 to 10 atoms and the heteroatom(s) beingselected from any of S, N and O and wherein the heteroaryl mayoptionally and independently be substituted by 1 or 2 substituents Ywherein each Y is as defined in claim
 1. 3. A compound according toclaim 2, wherein R¹ is (i) phenyl optionally and independently besubstituted by 1 or 2 substituents Y where each Y is as defined in claim1; (ii) naphthyl optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined in claim 1; (iii) heteroarylhaving from 5 to 10 atoms and the heteroatom being selected from any ofS, N and O; and wherein the aryl and heteroaryl may optionally andindependently be substituted by 1 or 2 substituents Y where each Y is asdefined in claim 1; Q is

 wherein R² is (i) phenyl optionally and independently be substituted by1 or 2 substituents Y where each Y is as defined in claim 1; or (ii)naphthyl optionally and independently be substituted by 1 or 2substituents Y where each Y is as defined in claim
 1. 4. A compoundaccording to any of the previous claims, which compound is anyoneselected from


5. A compound according to any one of the preceding claims, wherein saidcompound is in the form of a hydrochloride, sulfate, tartrate or citratesalt.
 6. A compound according to any one of claims 1-4, wherein saidcompound is isotopically labeled.
 7. A pharmaceutical compositioncomprising a compound of formula I according to claim 1 as an activeingredient, together with a pharmacologically and pharmaceuticallyacceptable carrier.
 8. A method for the treatment of a patient for pain,wherein an effective amount of a compound of formula I according toclaim 1 is administered to said patient.
 9. A method for the treatmentof a patient for a gastrointestinal disorder, wherein an effectiveamount of a compound of formula I according to claim 1, is administeredto said patient.
 10. A method for the treatment of a patient for aspinal injury, wherein an effective amount of a compound of formula Iaccording to claim 1, is administered to said patient.